The long term objective of this proposal is to understand the molecular mechanisms underlying alphabeta and gammadelta T cell development, to gain insight into the alphabeta versus gammadelta lineage decision making process and to understand the molecular differences between the two T cell lineages. The mouse T cell antigen receptor (TCR) alpha/delta locus gene regulation is an ideal and unique system to study for this purpose because the genes encoding the TCR delta chain are "nested" inside the TCR alpha genes but each is activated differentially during development in two different T cell lineages (alphabeta and gammadelta T cells). Hence special regulation must exist to assemble and to express the final rearranged product of the physically separate V, D, J and C (variable, diversity, joining and constant) gene segments of each TCR chain. Participation of TCR enhancers, silencers and promoters are crucial for this process. In this proposal, the TCR alpha enhancer and V gene segment promoters will be analyzed in great detail to understand how alphabeta T cell lineage specificity is achieved during T cell development and to identify as well as to characterize the proteins involved in the alphabeta T cell specific gene regulation. Several V alpha promoters will be assessed using gene transfection experiments into several alphabeta and gammadelta T cell lines. Molecules regulating differential expression of these Valpha promoters in the 2 T cell lineages will be analyzed using gel mobility shift and in vitro footprinting analysis. Mutagenesis of protein binding sites and functional analysis of the mutated V alpha promoter driving a reporter gene will then be performed to correlate protein binding activities with the V alpha promoter function. Similar studies using gel mobility shift and in vitro footprinting analysis will also be carried out for the TCR alpha enhancer to look for enhancer binding proteins. Their tissue distribution and developmental expression in normal tissues and cell lines will be assessed and the generality of these alpha enhancer binding proteins in regulating other T cell specific genes will be studied based on sequence homologies, competitive binding and in vitro mutagenesis of the relevant transcriptional elements. Protein/DNA interaction of the TCR alpha enhancer during development will also be studied using a polymerase chain reaction (PCR)-based in vivo footprinting analysis. Finally, a developmentally interesting TCR alpha enhancer binding protein will be characterized using biochemical means, and an expression cloning strategy will be followed in an attempt to isolate a gene encoding one of these T cell specific alpha enhancer binding proteins. As several oncogenes are translocated to the TCR alpha/delta locus in many of the acute and chronic leukemia T cells, these studies will have important significance to our basic understanding of the vertebrate immune response and the mechanisms of T cell tumorigenesis.